Myoblast fusion is a fundamental process for proper skeletal muscle formation during development and regeneration. Despite the importance of myoblast fusion for muscle formation, the mechanisms that govern this process are not fully understood. Elucidation of fusion mechanisms is a critical step for understanding muscle development and to develop new therapeutic strategies to augment skeletal muscle disease. We recently discovered a muscle-specific protein, named myomaker, that localizes to the plasma membrane of myoblasts. Genetic deletion of myomaker during development and adult regeneration renders myoblasts fusion incompetent, which results in a dramatic inability to form skeletal muscle. Moreover, expression of myomaker in cells that normally do not express this protein causes their fusion with muscle cells. While myomaker is a central component for myoblast fusion, the regulatory mechanisms that govern its activity and the biochemical function of myomaker remain unknown. We have identified the regions of myomaker that are critical for its function and the proteins that interact with myomaker to potentially regulate its activity. In his project we will: 1) understand the function of myomaker within the fusion process 2) identify effectors of myomaker activity 3) evaluate the optimal cell type for heterologous fusion and myogenic reprogramming. We will use genetic loss-of- and gain-of-function approaches in vivo to investigate the role of myomaker during fusion. Extensive molecular approaches within our myomaker-based heterologous fusion system are proposed to identify the mechanisms that govern myomaker action. The ability of myomaker to drive fusion of heterologous cells with muscle cells will serve as the foundation to repair diseased skeletal muscle. We will use cell transplantation experiments to assess fusion and subsequent reprogramming of non-muscle cells with normal and diseased muscle. Successful completion of these aims will provide insight into the mechanisms of myoblast fusion by revealing how myomaker is regulated to induce membrane merger. Finally, these studies will have significant implications for a myomaker-mediated cell based strategy to rescue genetic muscle diseases.